US9012147B2 - Method of enzymatic gene chip detection kit - Google Patents
Method of enzymatic gene chip detection kit Download PDFInfo
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- US9012147B2 US9012147B2 US13/906,890 US201313906890A US9012147B2 US 9012147 B2 US9012147 B2 US 9012147B2 US 201313906890 A US201313906890 A US 201313906890A US 9012147 B2 US9012147 B2 US 9012147B2
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- gene chip
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2521/00—Reaction characterised by the enzymatic activity
- C12Q2521/50—Other enzymatic activities
- C12Q2521/537—Protease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/125—Specific component of sample, medium or buffer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/50—Detection characterised by immobilisation to a surface
- C12Q2565/507—Detection characterised by immobilisation to a surface characterised by the density of the capture oligonucleotide
Definitions
- the present invention relates to detection with a gene chip; more particularly, relates to using a gene chip having 17 agents for clinical direct detection on blood without magnification.
- RT-PCR reverse transcription polymerase chain reaction
- real-time PCR PCR-time PCR
- First problem pollution which caused false positive on detection.
- Second one is related to RT-PCR, where, on comparing different samples, it is hard to control efficiency of sequence magnification.
- Third one is interference on annearling between primers, where, on detecting gene groups, PCR-related technologies will take time, become complex and cost high.
- Another prior art is a gene chip of weighted chemiluminescent membrane array (WCHMA). Abnormal situation of target K-ras over a target therapeutic drug for a lung cancer patient is analyzed.
- WCHMA weighted chemiluminescent membrane array
- the main purpose of the present invention is to use a gene chip for clinical direct detection on blood without magnification to provide high sensitivity and convenience at the same time.
- the present invention is a method of an enzymatic gene chip detection kit, comprising steps of:
- oligonucleotide segment for 384 ⁇ 576 min to obtain a hybridization solution, where an oligonucleotide segment is selected; the oligonucleotide segment comprises a plurality of target genes; two housekeeping genes are obtained to control reaction quality; the housekeeping genes fixes the oligonucleotide segment on the gene chip to obtain a specific sequence covered on the gene chip; and
- FIG. 1 is the flow view showing the preferred embodiment according to the present invention.
- FIG. 1 is a flow view showing a preferred embodiment according to the present invention.
- the present invention is a method of an enzymatic gene chip detection kit.
- the present invention uses an enzymatic gene chip detection kit, comprising a gene chip and a set of gene chip reagents.
- the gene chip has two housekeeping genes for controlling quality of reactions of the gene chip.
- the set of gene chip reagents comprises all reagents required in a flow of the reactions of the gene chip.
- the flow of the reactions of the gene chip comprises RNA extraction, cDNA synthesis, probe labeling, hybridization and signal detection.
- the gene chip requires 17 reagents for the reactions. After the reactions, expressions of the housekeeping genes are observed.
- the present invention comprises the following steps:
- RNA extraction 11 A sample is evenly mixed with 6 mAU of a protease K solution. Then, a lysis solution is added to be evenly mixed in.
- the lysis solution comprises 200 millimoles (mM) of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 400 mM of sodium chloride (NaCl) and 1% of octyl phenoxy polyethoxy (Triton X-100).
- a magnetic bead solution is added to be evenly mixed in, which is a solution of 25 mg/ml of hydroxyl-silicon dioxide magnetic bead.
- the mixed solution is evenly mixed with a binding solution consisting 12.5% of polyethylene glycol (PEG) and 2 moles (M) of NaCl.
- a binding solution consisting 12.5% of polyethylene glycol (PEG) and 2 moles (M) of NaCl.
- PEG polyethylene glycol
- M moles
- a washing solution consisting 70% of ethanol, 1% of glycerin and 100mM of ammonium acetate, is evenly mixed in.
- the magnetic beads are fixed by a magnet again for removing a reacting solution thus obtained.
- RNA solution consisting 20 mM of tris-hydrogen chloride (Tris-HCl) and 1 mM of ethylenediaminetetraacetic acid (EDTA), is evenly mixed in. After the mixed solution is reacted at a temperature of 70° C. for 10 min, the magnetic beads are fixed by a magnet for collecting an RNA solution thus obtained.
- Tris-HCl tris-hydrogen chloride
- EDTA ethylenediaminetetraacetic acid
- cDNA synthesis 12 After putting the RNA solution for reaction at a temperature of 70° C. for 10 min, a primer solution, consisting 300 nanograms per microliter (ng/ul) of oligo-dT primer and 100 ng/ul of random primer, is evenly mixed in. After putting the mixed solution for reaction at a temperature of 0° C. for 2 min, a reverse transcription solution, consisting 250 mM of Tris-HCl, 375 mM of potassium chloride (KCl), 15 mM of magnesium chloride (MgCl) and 100 mM of dithiothreitol (DTT), is evenly mixed in.
- a reverse transcription solution consisting 250 mM of Tris-HCl, 375 mM of potassium chloride (KCl), 15 mM of magnesium chloride (MgCl) and 100 mM of dithiothreitol (DTT)
- a reverse transcriptase solution consisting 200 units of reverse transcriptase MLV and 25 units of ribonuclease inhibitor (RNases inhibitor), is evenly mixed in. After putting the mixed solution for reaction at a temperature of 42° C. for 120 min, a cDNA solution is obtained.
- RNases inhibitor ribonuclease inhibitor
- Probe labeling 13 After putting the cDNA solution for reaction at a temperature of 95° C. for 5 min, the cDNA solution is reacted at a temperature of 0° C. for 2 min and, then, is added with a labeling solution evenly mixed in, where the labeling solution comprises 80 nanograms per milliliter (ng/ml) of random primer, 2 units per microliter (U/ul) of Klen Taq polymerase, 1 mM of deoxyadenosine triphosphate (dATP), 1 mM of deoxycytidine triphosphate (dCTP), 1 mM of deoxyguanosine triphosphate (dGTP), 0.6 mM of deoxythymidine triphosphate (dTTP), 0.4 mM of biotin-deoxyuridine triphosphate (biotin-dUTP), 20% of Klen Taq buffer and 20% of glycerin. After putting the mixed solution for reaction at a temperature of 42° C. for 240 min, a
- Hybridization 14 A gene chip is put into a reaction chamber.
- a hybridization solution consisting 10% of polyethylene glycol (PEG), 10% of sodium dodecyl sulfate (SDS) and 2 moles (M) of sodium chloride (NaCl), is evenly mixed in.
- the probe solution is reacted at a temperature of 95° C. for 5 min and continuous reaction at a temperature of 0° C. for 2 min. After putting the mixed solution for reaction at a temperature of 42° C. for 10 min, the probe solution is added into the reaction chamber. After putting the mixed solution for reaction at a temperature of 42° C. for 480 min, a hybridization solution is obtained.
- Signal detection 15 The hybridization solution is removed to leave the gene chip in the reaction chamber.
- a first washing solution consisting 0.5% of SDS and 0.5 ⁇ of saline sodium citrate (SSC), is added and is removed after 5 min of reaction. In the same way, the first washing solution and is added and is removed again.
- a second washing solution of 2 ⁇ of SSC is added and is removed after 5 min of reaction at a temperature of 42° C. In the same way, the second washing solution is added and is removed again.
- a filling solution consisting 1.5% of blocking buffer, is added and removed after 30 min of reaction.
- An antibody solution consisting 0.7 ug/ml of alkaline phosphatase-conjugated IgG anti-biotin, is added and removed after 90 min of reaction.
- a third washing solution consisting 1% of SDS, 1% of octyl phenoxy polyethoxy and 1% of glycerin, is added and removed after 5 min of reaction. In the same way, the third washing solution is added and is removed again.
- a chromogen solution consisting 1 ⁇ of 5-bromo-4-chloro-3-indolyl-phosphate/p-nitro-blue tetrazolium chloride (BCIP/NBT), is added for reaction at a temperature of 42° C. until signals are generated. Then, water is added for washing until signals stop generating. A detection result is finally obtained after drying.
- the gene chip is a material coated with a nylon membrane or a thermoplastic composite;
- the thermoplastic composite can be polypropylene (PP) or polymethyl methacrylate (PMMA).
- PP polypropylene
- PMMA polymethyl methacrylate
- an oligonucleotide segment is selected; the oligonucleotide segment comprises a plurality of target genes; two housekeeping genes are used to control reaction quality; and, the housekeeping genes fixes the oligonucleotide segment on the gene chip to form a specific sequence covered on the gene chip.
- the oligonucleotide segment is a synthetic oligonucleotide consisting in a solution; the solution has a density of 10 ⁇ 200 mM; the oligonucleotide segment has a length of 40 ⁇ 60 bases; specific sequence of each of the target genes are complement to the those of the other target genes; more than 30 normal liters (NL) of the solution of synthetic oligonucleotide is distributed on the gene chip to duplicate and arrange the oligonucleotide segment in a planned way; and, after being dried, the oligonucleotide segment is radiated by ultraviolet (UV) to be solidified.
- UV ultraviolet
- the present invention can be used in clinical tests, where direct test on blood is possible without magnification.
- gene chip gene expressions in the blood are observed with high sensitivity and convenience.
- the present invention is a method of an enzymatic gene chip detection kit, where direct test on blood is possible without magnification; and, with a gene chip fabricated according to the present invention, gene expressions in the blood are observed with high sensitivity and convenience.
Abstract
Description
Claims (20)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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TW101144416 | 2012-11-28 | ||
TW101144416A TWI509074B (en) | 2012-11-28 | 2012-11-28 | Enzyme Gene Chip Detection Reagent and Its Detection |
TW101144416A | 2012-11-28 |
Publications (2)
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US20140147841A1 US20140147841A1 (en) | 2014-05-29 |
US9012147B2 true US9012147B2 (en) | 2015-04-21 |
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US13/906,890 Expired - Fee Related US9012147B2 (en) | 2012-11-28 | 2013-05-31 | Method of enzymatic gene chip detection kit |
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US (1) | US9012147B2 (en) |
CN (1) | CN103849679B (en) |
TW (1) | TWI509074B (en) |
Families Citing this family (8)
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US10252500B2 (en) * | 2014-10-02 | 2019-04-09 | Solutia Inc. | Multiple layer interlayer resisting defect formation |
US9809010B2 (en) * | 2014-10-15 | 2017-11-07 | Solutia Inc. | Multilayer interlayer having sound damping properties over a broad temperature range |
TWI703217B (en) * | 2016-04-22 | 2020-09-01 | 康雋國際生物科技股份有限公司 | High-performance mRNA labeling detection method |
TWI732827B (en) * | 2017-02-24 | 2021-07-11 | 大陸商蘇州翊準基因科技有限公司 | Detection method of high-efficiency mRNA labeling detection reagent set |
TWM567771U (en) * | 2017-09-08 | 2018-10-01 | 翊准國際生物科技股份有限公司 | High-performance mRNA marker auto-analysis device |
CN108531548A (en) * | 2018-04-09 | 2018-09-14 | 上海芯超生物科技有限公司 | A kind of cell cDNA chip and its preparation method and application |
CN110628762A (en) * | 2019-10-14 | 2019-12-31 | 杭州同创越诚基因科技有限公司 | Nucleic acid extraction method based on nano magnetic beads and application |
CN112048401B (en) * | 2020-08-28 | 2022-05-17 | 上海符贝基因科技有限公司 | Micro-fluidic chip cleaning agent and method thereof |
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WO2007011412A2 (en) * | 2004-11-05 | 2007-01-25 | The Government Of The United States Of America, As Represented By The Secretary Of The Navy | Diagnosis and prognosis of infectious diesease clinical phenotypes and other physiologic states using host gene expresion biomarkers in blood |
CN101665785B (en) * | 2009-09-24 | 2011-02-16 | 戴立忠 | Method for extracting and purifying nucleic acid from samples by magnetic beads |
TWI402502B (en) * | 2010-05-13 | 2013-07-21 | Fooyin University Hospital | Genome detection system |
CN102191339B (en) * | 2011-03-30 | 2014-01-29 | 珠海出入境检验检疫局检验检疫技术中心 | Preparation method and detection method for gene chip |
CN102229925B (en) * | 2011-05-13 | 2013-04-17 | 薛昱 | Enhanced magnetic-bead-based nucleic acid extraction method |
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2012
- 2012-11-28 TW TW101144416A patent/TWI509074B/en active
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2013
- 2013-05-31 US US13/906,890 patent/US9012147B2/en not_active Expired - Fee Related
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Publication number | Publication date |
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CN103849679B (en) | 2017-04-26 |
US20140147841A1 (en) | 2014-05-29 |
CN103849679A (en) | 2014-06-11 |
TW201420767A (en) | 2014-06-01 |
TWI509074B (en) | 2015-11-21 |
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